Determination of increased amounts of renin in body fluids

ABSTRACT

Inhibition of the action of renin by pepstatin both in vivo and in vitro is useful in determining the presence of reninassociated hypertension. Comparative pressor effects in mammals upon administration of renin-containing systems, with and without added pepstatin, can be employed for the determination of the amounts of renin in said systems.

United States Patent [1 1 Miller Mar. 25, 1975 DETERMINATION OFINCREASED Umezawa. J. of Antibiotics, Vol. 23, May 1970, pp.

AMOUNTS 0F RESIN IN BODY FLUIDS 259461 [75] Inventor: Richard P. Miller,Zionsville, Ind. of Annbloncs 23 1970 [73] Assignee: Eli Lilly andCompany, Indianapolis, Chem. Abs., Vol. 74, No. 752010 (1971).

Ind.

[22] Filed. May 21 1973 Primary Examiner-Albert T. Meyers AssistantExaminer-A. P. Fagelson PP ,574 Attorney, Agent, or Firm]ames L. Rowe;Everet F.

Related U.S. Application Data Smlth [62] Ser. No. 226.744, Feb. 16,1972, Pat. No. ABSTRACT Inhibition of the action of renin by pepstatinboth in [52] U.S. Cl. 424/9, 424/94 vii/0 and in vitro is useful indetermining the presence 51 rm. Cl ..G01n 33/00, GOln 33/16, ofrenin-associated hypertension Comparative Pressor A 1 27/ effects inmammals upon administration of renin- [58] Field of Search 424/9, 94Containing Systems, with and Without added p p can be employed for thedetermination of the amounts 5 References Cited of renin in saidsystems.

OTHER PUBLICATIONS Gross. Seience, Vol. 175, Feb. 11, 1972 pg. 656.

1 Claim, 4 Drawing Figures FATENTEU 3 sum 2 or 1 BLOOD PRESSURE NORMALRAT mmHg B mmHg A m pi -I'M f SALINE VOLUME CONTROL PEPSTATIN 260 pg/kqI l I l l l l J l I 125 PEPSTATIN 200 pq/kq STARTED RENIN INFUSION T 115MINUTES (continuous record) mmHg PEPSTATIN zoo pq/kg l l l l l L l l l I12 13 14 15 16 17 I8 19 20 21 22 I INFUSION STOPPED MINUTES (continuousrecord) Fig. 2

P/JENi'EU 1.8731581 sum u o 1 PEPSTATIN DQSE RESPONSE CURVE 5 4 3 2 EI Vwmswmmxm 55 5 E wmfimuwm PEPSTATiN (pg/kg) Fig. 4

DETERMINATION OF INCREASED AMOUNTS OF RESIN IN BODY FLUIDS This is adivision of application Ser. No. 226,744, filed Feb. 16, 1972, now US.Pat. No. 3,784,686.

BACKGROUND OF THE INVENTION According to Goodman and Gilman, ThePharmacological Basis of Therapeutics, page 663 et seq, 4th Ed., (NewYork: The Macmillan Company, 1970), renin was first isolated from crudesaline extracts of the kidney in 1898. The substance had a markedpressor effect but was of little interest until 1934 when Goldblatt andhis colleagues were able to produce hypertension in dogs by constrictingthe renal artery. Later investigators were able to find renin in therenal venous blood of these Goldblatt dogs. Further investigation hasshown that renin is not itself a pressor substance but is an enzyme thatacts on a substance in the plasma known as renin-substrate to produceangiotensin I which is converted to angiotensin II, a powerful pressorsubstance. It is important for the differential diagnosis ofhypertension to know whether the disease is associated with increasedplasma levels of renin. The determination of renin in body fluidsincluding arterial blood is complicated and time consuming, frequentlyrequiring more than 24 hours for a determination.

Pepstatin is an elaboration product of numerous species ofactinomycetes. Its isolation is described in J. Am tibia, 23 259 (1960).Pepstatin was discovered by Unezawa and co-workers. Its structure isdescribed in J. Antibz'o. 23, 263 (1970). Pepstatin inhibits the actionof pepsin when added to preparations of that enzyme. Pepstatin alsoprevents ulceration of the pylorus in Shav rats.

It is an object of this invention to provide a method of determiningrenin both in vitro and in vivo which is both accurate and rapid.

SUMMARY OF THE INVENTION In fulfillment of the above and other objects,this invention provides a method for inhibiting the action of reninwhich comprises adding pepstatin to the renincontaining system thusdecreasing the activity of renin present. The renin originally presentcan then be measured usually by determining the pressor effect of therenin-containing system with and without added pepstatin.

When the inhibition of renin by pepstatin is used to determine theamount of renin in a body fluid, both in vivo and in vitrodeterminations can be used.

In the in vivo assay for renin, pepstatin is injected into the animal atsuitable dosage levels, preferably above 50 ,ug./kg. When injected inthis manner, pepsta' tin produces a transitory fall in blood pressure inGoldblatt rats or in normal rats in whom the blood pressure has beenelevated by a continuous infusion of renin. Other mammals havingelevated blood pressure associated with above-normal circulating reninlevels will also suffer a brief fall in blood pressure when injectedwith suitable levels of pepstatin. Such a fall in blood pressure is thusindicative of the presence of renin.

With one in vitro assay, the body fluid, usually plasma, can beincubated with pepstatin and, after deproteinization, the amountofpressor peptide produced is measured in the nephrectomizedpentolinium-treated rat. In a second in vitro assay, the plasma is mixedwith a suitable amount of pepstatin, and the mixture injected into atest animal. The difference in pressor response with and without addedpepstatin is a measure of the renin content of the plasma.

It has also been demonstrated that the effect of pepstatin was specificfor renin since infusion of a pressor substance completely unrelated torenin gave a sustained rise in blood pressure not affected by theinjection of pepstatin. This experiment was carried out in a normal ratin which the pressor substance was infused and pepstatin injected atintervals.

As previously stated, the process of this invention is useful indetermining whether hypertension occurring spontaneously in mammals isassociated with increased renin levels. Employing my novel in vivoprocess, it is possible to make a determination of the presence ofelevated renin levels in a hypertensive mammal within 5 minutes. About 1minute after pepstatin has been injected into the hypertensive mammal,the blood pressure begins to fall and will return to the previouslyelevated level by the end of5 minutes if no more pepstatin is injected.The in vitro assay takes somewhat longer, but is nevertheless a rapidassay, requiring only a previously prepared animal into whom therenin-containing body fluid can be injected. Injecting therenincontaining material which has been mixed with pepstatin yields alower increase in blood pressure in a normotensive rat as compared withuntreated material. Similarly, injection of the product of theincubation of a remin-containing body fluid with pepstatin into apentolinium-treated, nephrectomized rat gives a lower pressor responsethan incubated material to which pepstatin has not been added.

Another use of our novel process is the reduction in blood pressure ofactively hypertensive patents associated with increased plasma reninlevels (B.P.=280 mm/I-Ig, for instance) in clinical situations such ascerebral vascular hemorrhage secondary toincreased blood pressure wherethe blood pressure must be reduced very rapidly, at least for shortperiods of time.

This invention is further illustrated by the following specificexamples.

EXAMPLE 1 In vitro inhibition of renin by pepstatin Renin assaying at 7to 8 GU/mg. was obtained from a commercial source. The renin wasincubated with an excess of crude bovine renin substrate prepared by themethod of Skeggs et al J. Exp. Med., 118 73 (1963), at pH=6.5 and at atemperature of about 37C. for 2 hours. The enzymatic reaction wasstopped by heating the reaction mixture at about C. for about 10minutes. The denatured proteins produced during this heating wereremoved by centrifugation. The supernatant liquid was then assayed forpressor activity in a nephrectomized, pentolinium-treated rat, usingangiotensin as the standard. The same enzymatic assay was carried outwith different levels of pepstatin being added to the incubationmixture. The inhibition curve shown in FIG. 1 was obtained from such aseries of incubations. From the curve, the EC (effective concentrationgiving a 50 percent inhibition of renin for the pepstatin-renin mixture)was determined to be 0.32 X 10 molar. In a similar experiment, in whichdialyzed plasma from a nephrectomized cat was used as a substrate,inhibition of renin was obtained by the addition of pepstatin even up toaddition times as late as 40 minutes after the start of a 1 hourincubation period.

EXAMPLE 2 In vivo inhibition of renin activity by pepstatin A pithednephrectomized cat was injected with 0.1 GU of renin. The consequentrise in blood pressure was about 21 mm. of mercury. When pepstatin wasinjected intravenously into the same cat, the later intravenousinjection of renin gave a rise in blood pressure of only mm. of mercury.The renin had to be injected within 2 circulation times after theinjection of the pepstatin, both being given by the intravenous route,in order for the above lessened pressor effect to be found.

EXAMPLE 3 In vivo inhibition of the action of renin by pepstatinAnesthetized rats were prepared so that their blood pressure could becontinuously recorded via the carotid artery while the animals werebeing maintained with a positive pressure pump. The right femoral veinwas cannulated for infusion and the left femoral vein cannulated forinjection. Pepstatin injected into normal rats prepared as above atdosage levels up to 260 pg/kg. had no effect on blood pressure. When theblood pressure was elevated, however, by -25 mm. of mercury by thecontinuous infusion of renin into the right femoral vein, the injectionof 200 pg/kg. of pepstatin into the left femoral vein reduced the bloodpressure to normal in less than 1 minute as shown in the continuingcurve in FIG. 2. In this type of preparation, if the infusion of reninwas stopped immediately after the injection of pepstatin, the elevatedblood pressure produced by the renin infusion returned to normal range 4times faster than in the absence of pepstatin. These results arerecorded in FIG. 2.

EXAMPLE 4 In vivo hypotensive activity of pepstatin Chronicallyhypertensive Goldblatt rats were prepared in the same way as the normalrats in Example 2. A definite hypotensive response was obtained by in-The inhibition of the action of renin by pepstatin is a competitiveinhibition as shown by a Lineweaver- Burke plot of the kinetic data.Pepstatin has no effect on the blood pressure of normal rats. Thus,pepstatin is an ideal candidate for use as a clinical diagnostic toolfor determining whether a given hypertensive patient has arenin-associated component in his or her hypertension. In employing theprocess of this invention as a diagnostic aid, the in vivo determinationis probably preferable to the in vitro determination in that the formerrequires only the injection of pepstatin into patients having anelevated blood pressure with a continuous measurement of the bloodpressure. In such a determination, an immediate transitory fall of bloodpressure would be indicative of a renin-associated component in thehypertensive state. In carrying out the in vivo assay for the presenceof renin, the dosages of pepstatin which are employed can vary from50-1600 pig/kg. of mammalian body weight. The preferred amount ofpepstatin employed is 200 pg/kg. of mammalian body weight. The in vitrodetermination, however, is also relatively easy and is carried out bytaking blood from the hypertensive patient, incubating it with reninsubstrate with and without the addition of pepstatin and then measuringthe amount of pressor peptides produced by the difference in effect onblood pressure in normotensive rats. A lowered blood pressure in thepresence of pepstatin would be indicative of a reninassociatedhypertension.

For intravenous injection into mammals, pepstatin is convenientlydissolved in isotonic phosphate-buffered saline at pH=7.3 or thereabout.The concentration of pepstatin is usually in the range l00-125 pg/ml.

I claim:

1. A method of determining increased amounts of renin in mammalian bodyfluids wherein the body fluid is incubated with renin substrate andpepstatin, denatured proteins are removed and the supernate of saidincubation is parenterally administered to a nephrectomized, pentoliniumtreated rat, with the lowered pressor effect of this incubation mixturebeing a measure of the amount of renin in said body fluid when comparedwith the pressor effect of adminstration of the supernate of theincubation of the same body fluid and renin substrate without theaddition of pepstatin.

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UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTIONPATENT N0. 1 3, 73,

DATED March 25, 1975 INVENTOR(S) Richard P. Miller It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below: 0 In the title on page 1,"RESIN" should be BENIN Column 1, line 2, in the title "RESIN" should beBENIN --t.

Column 1, line 55, "Shav" should be Shay Sugncd and Sealed thlsFourteenth Day Of September 1976 [SEAL] Q Arrest:

RUTH c. MASON c. MARSHALL DANN Arresring Officer Commissioner uj'Parenrsand Trademarks

1. A METHOD OF DTERMINING INCREASED AMOUNT OF RENIN IN MAMMALINA BODYFLUIDS WHEREIN THE BODY FLUID IS INCUBATED WITH RENIN SUBSTRATE ANDPREPSTATIN, DENATURED PROTEINS ARE REMOVED AND THE SUPERNATE OF SAIDINCUBATION IS PARENTERALLY ADMINSTERED TO A NEPHREETOMIZED PENTOLINIUMTREATED RAT, WITH THE LOWERED PRESSOR EFFECT OF THIS INCUBATION MIXTUREBEING A MEASURE OF THE AMOUNT OF RENIN IN SAID BODY FLUID WHEN COMPAREDWITH THE PRESSOR EFFECT OF ADMINSTRATION OF THE SUPERNATE OF THEINCUBATION OF THE SAME BODY FLUID AND RENIN SUBSTRATE WITHOUT THEADDITION OF PEPSTATIN.